Cryptococcus neoformans is a pathogenic yeast which belongs to Basidiomycetes, a taxonomic Class phylogenetically remote from the rest of the pathogenic yeasts (Ascomycetes). C. neoformans causes fatal meningoencephalitis primarily in patients with T- lymphocyte dysfunction and is the only species in the genus Cryptococcus that has optimum growth at temperatures higher than 30C. Although extensive studies on the molecular basis of cell cycle and morphogenesis have been carried out in ascomycetous yeasts, such studies have not been conducted in C. neoformans. This project was initiated to study the molecular genetics of cell cycle associated genes in C. neoformans. In 1999, we reported a strain (B-4551) isolated from a chronic granulomatous lesion that exhibited aberrant cell morphology at 35 C and was unable to multiply at 37 C. This strain fails to cause systemic infection in mice. Last year we identified the genetic defect in this strain and isolated the CCN1 gene which could complement the mutant phenotype (Ts) in vitro and allows the mutant to cause systemic fatal infection in mice. The CCN1 gene encodes a protein containing 16 tetratricopeptide repeats (TPR) of 34 amino acids each that shows high homology with two essential genes, CRN of Drosophila and CLF1 of yeast. The CRN is involved in neurogenesis and the CLF1 gene encodes a spliceosome assembly protein. We have shown that the S. cerevisiae CLF1 gene complements the Ts phenotype of B-4551 but the CCN1 cDNA failed to rescue the null allele of clf1. This year we further analyzed the possible association of CCN1 with splicing using three multiple intron possessing genes: GPD, RHO1 and CCN1. Northern analysis as well as RTPCR failed to detect accumulation of pre-mRNA when exposed to restricted temperatures. However, FACS analysis indicated that the CCN1 gene is associated with cell cycle progression. The population in G2 phase was found to be drastically reduced when the ccn1 mutant was exposed to 37 C.